Foram sintetizadas bisporfirinas, em que as unidades de porfirina encontram-se ligadas às posições 4,4\' da 2,2\'bipiridina através da formação de ligações amídicas, pela reação entre uma aminofenil porfirina monofuncionalizada (NH2PTriPP) e a 4,4\'-dicloreto de ácido bipiridina (DACBipy). Para a obtenção da NH2PTriPP foi necessário um estudo preliminar das condições de reação de síntese da molécula precursora da NH2PTriPP, a correspondente mono-nitrofenil porfirina (NO2PTriPP). A NO2PTriPP foi obtida com rendimentos da ordem de 60% em reações de nitração da TPP em ácido trifluoracético a temperatura ambiente, excessos de duas vezes de nitrito de sódio e tempos de reação não maiores que um minuto. Desta forma são obtidas apenas a mononitroporfirina e TPP que não reagiu sem a presença de outros produtos nitrados. Nestas condições se obtém grande reprodutibilidade e a mistura resultante de NO2PTriPP e TPP pode ser diretamente reduzida por cloreto de estanho para a obtenção da NH2PTriPP sem necessidade da separação da TPP. Após uma série de testes foi obtida a bisporfirina-bipiridina (BisPBipy) pela reação entre a NH2PTriPP e a DACBipy, sendo aspectos importantes nesta reação a obtenção do cloreto de ácido da bipiridina (DACBipy) in situ e utilização de excessos de aminoporfirina, ao invés de relações estequiométricas como é usual para este tipo de reação. Desta forma se evita a conversão do cloreto de ácido no respectivo ácido carboxílico que apresenta menor solubilidade e reatividade. Uma vez obtida a bisporfirina base livre foi possível sintetizar a respectiva bisporfirina metalada com íons de Zn(II) nas unidades de porlitina(BisZnPBipy) pela reação com cloreto de zinco. Os espectros de absorção da BisPBipy e BisZnPBipy apresentam perfil espectral característicos das porfirinas base livre e metaloporfirina, sendo os máximos de absorção tanto da banda Soret como das bandas Q praticamente coincidentes com os máximos de absorção da TPP e ZnTPP. As absortividades molares, entretanto, são duas vezes maiores, do que a TPP e ZnTPP, indicando a existência de duas unidades cromofóricas porfirínicas. Assim como observado para as propriedades de absorção UV-Vis, existe uma grande similaridade nas propriedades de emissão da BisPBipy e BisZnBipy com as moléculas modelo TPP e ZnTPP, respectivamente. A BisPBipy mantém o espectro de emissão de \"imagem especular reversa\" da TPP, indicando que a ligação a unidade de bipiridina não altera a geometria do estado excitado. Tanto a BisPBipy como a BisZnPBipy apresentam rendimentos quânticos de emissão de fluorescência similares a TPP e ZnTPP, indicando que para uma mesma fração de luz absorvida tem-se uma mesma emissão de fluorescência por unidade porfirínica presente nas bisporfirinas. Portanto as propriedades de absorção e emissão indicam que as unidades de porfirina atuam de forma independente não sendo influências pela ligação a unidade de bipiridina. Foi investigada a interação entre as bisporfirinas-bipiridinas e os íons Zn2+, Fe2+, Ni2+, Cu2+ e Co2+ em THF. Através de espectroscopia eletrônica UV-Vis e emissão de fluorescência foi possível constatar que ocorre interação entre as bisporfirinas-bipiridinas e os íons Ni2+, Cu2+ e Co2+. A presença destes íons metálicos em soluções das bisporfirinas-bipiridinas origina apenas pequenas alterações na intensidade da banda Soret; as bandas Q permanecem inalteradas. Este resultado é decorrente da interação dos íons metálicos com a unidade de bipiridina destas bisporfirinas, sendo que as alterações espectrais devidas a complexação ocorrem nas bandas centradas no ligante bipiridina que apresentam transições no ultravioleta, não interferindo apreciavelmente na região espectral das porfirinas. Contrariamente ao que foi observado nos espectros de absorção a presença de íons metálicos influencia de forma significativa a intensidade de fluorescência das bisporfirinas-bipiridinas investigadas, podendo ser observada a supressão de fluorescência em função do aumento da concentração dos íons metálicos. Utilizando os dados de emissão de fluorescência foi possível determinar a estequiometria dos complexos formados entre as bisporfirinas-bipiridinas e os íons metálicos. Co2+ e Cu2+ formam complexos de estequiometria 1:1, enquanto que Ni2+ forma complexos de estequiometria 2:1. A análise dos plots do tipo Stern-Volmer para a supressão de fluorescência das bisporfirinas-bipiridinas mostra um perfil exponencial típico de um processo de supressão não difusional. As constantes de Stern-Volmer aparentes (KSVap) mostram que não existe uma diferença significativa na eficiência de supressão independentemente da bisporfirina analisada ou do íon metálico supressor, sendo obtidos valores de KSVap de aproximadamente 5x105 M-1. Também foi realizado um estudo da interação das bisporfirinas-bipiridinas com o complexo [Eu(tta)3(H2O)2, não sendo observada uma interação apreciável nas condições normais de titulação, indicando que substituição das moléculas de água coordenada no complexo [Eu(tta)3(H2O)2 pela bipiridina não é favorecida em THF. Para as amostras submetidas a aquecimento é verificada uma supressão de fluorescência de aproximadamente 25-30%, indicando a ligação com a unidade bipiridina. Estas bisporfirinas se apresentam como moléculas promissoras no desenvolvimento de sistemas mais complexos contendo porfirinas devido a presença das unidades de bipiridina que possibilitam a formação de uma série de novas estruturas baseadas na coordenação a unidade de bipindina. Outra perspectiva é a elaboração bisporfirinas-bipiridinas, contendo outras metaloporfirinas como, por exemplo, de paládio, estanho, cobre, que também são luminescentes, porem com outras propriedades de estado excitado, resultando em sistemas de grande versatilidade. / In this work have been synthesized bisporphyrins held together by means of amide linkage at the 4,4\' position of 2,2-bipyridine, reacting the mono-functionalised aminophenyl-porphyrin (NH2PTriPP) and 4,4\'-diacid chloride bipyridine (DACBipy). A preliminary study was conducted for the establishment of the reaction conditions for the attainment of mono(nitrophenyl)-porphyrin (NO2PTriPP), the corresponding porphyrin precursor for the NH2PTriPP synthesis. The NO2PTriPP was obtained with yields in the range of 60% in nitration reactions of TPP in trifluoroacetic acid at room temperature, using twofold molar excess of sodium nitrite and period of reaction no longer than one minute. With these reaction conditions are achieved high reproducibility and the resulting mixture of NO2PTriPP and TPP can be directly reduced by tin chloride for obtaining NH2PTriPP without the need of TPP separation. After a series of trials the bisporphyrin-bipyridine (BisPBipy) was obtained reacting NH2PTriPP and DACBipy. Important features of this reaction conditions are the preparation in situ of the diacid chloride bipyridine and the use of a larger excess of aminoporphyrin than the usual for this kind of reaction. In this way, the formation of the corresponding carboxylic acid bipyridine is avoided. The correspondent Zn(II) bisporphyrin-bipyridine derivative (BisZnPBipy), was obtained reacting the free base BisPBipy with zinc chloride. The BisPBipy and BisZnPBipy show characteristic spectral profile of free base and metalloporphyrin spectra, and was observed a great similarity in the absorption maxima for the Soret and Q bands with the corresponding maxima of TPP and ZnTPP respectively. Otherwise the molar absorptivities are twofold higher, as an indication of the existence of two chromophoric units, as expected for bisporphyrins. As observed for the UV-Vis absorption spectra there are a great similarity in the emission properties of BisPBipy and BisZnPBipy and the models TPP and ZnTPP respectively. The \"reverse mirror image\" emission of TPP is also observed for BisPBipy, indicating that the porphyrin chromophore bounded to the bipyridine unit does not alter the excited state geometry. The fluorescence quantum yields for BisPBipy and BisZnPBipy are the same to the observed for TPP and ZnTPP, indicating that for the same fraction of absorbed light the same fluorescence is displayed per porphyrin unit in the bisporphyrin. It can be concluded from the absorption and emission properties that the porphyrin chromophore in these bisporphyrins behaves as independent units. The interaction between the bisporphyrins-bipyridines and Zn2+, Fe2+, Ni2+ , Cu2+ and Co2+ ions in THF was investigated by means of UV-Vis electronic absorption and fluorescence emission spectroscopies, the results show that the interaction occurs only with Ni2+ , Cu2+ e Co2+ without formation of any precipitate. The presence of these metal ions in the bisporphyrin-bipyridine solutions causes small decrease in the intensity of the Soret band; the Q bands remaining unaltered. These spectral features are due to the interaction at the bipyridine unit of these bisporphyrins, and the resulting spectral changes occur in the ligand-centered bipyridine bands showing absorption in the ultra-violet, not matching appreciably the porphyrin absorption spectrum region. The presence of metal ions deeply influences the fluorescence emission intensity of these bisporphyrins, being observed the fluorescence quenching as function of the increasing metal ion concentration. From the fluorescence emission data the stoichiometry of the complexes formed in solution has been estimated for Co2+ and Cu2+ as 1:1 and for Ni2+ as 2:1. The analysis of the Stern-Volmer plots shows a typical profile of a non diffusional quenching process. The apparent Stern-Volmer constants (KSVap) show no significant differences independently of the bisporphyrin or the metal ion employed, being obtained KSVap values of approximately 5x105 M-1. In addition, it was studied the interaction between the bisporphyrins-bipyridines and the [Eu(tta)3(H2O)2 complex, with no appreciable interaction occurring at normal titrating conditions, as an indicative of disfavoring process of the replacement of the coordinated water molecules by the bipyridine ligand in THF. For the samples submitted to heating a fluorescence quenching of 25-30% can be observed, indicating that under heating conditions the complex with the bipyridine is formed. These bisporphyrins are promising in the development of more complex systems containing porphyrins due the presence of the bipyridine units that enable the formation of a series of new structures based on the bipyridine coordination. Another perspective is regarded to the elaboration of bisporphyrins-bipyridine containing different metalloporphyrins, such as palladium, tin, copper, that show luminescence, but with different excited state properties than the zinc porphyrin ones, resulting in systems of great versatility.

Foram sintetizadas bisporfirinas, em que as unidades de porfirina encontram-se ligadas às posições 4,4\' da 2,2\'bipiridina através da formação de ligações amídicas, pela reação entre uma aminofenil porfirina monofuncionalizada (NH2PTriPP) e a 4,4\'-dicloreto de ácido bipiridina (DACBipy). Para a obtenção da NH2PTriPP foi necessário um estudo preliminar das condições de reação de síntese da molécula precursora da NH2PTriPP, a correspondente mono-nitrofenil porfirina (NO2PTriPP). A NO2PTriPP foi obtida com rendimentos da ordem de 60% em reações de nitração da TPP em ácido trifluoracético a temperatura ambiente, excessos de duas vezes de nitrito de sódio e tempos de reação não maiores que um minuto. Desta forma são obtidas apenas a mononitroporfirina e TPP que não reagiu sem a presença de outros produtos nitrados. Nestas condições se obtém grande reprodutibilidade e a mistura resultante de NO2PTriPP e TPP pode ser diretamente reduzida por cloreto de estanho para a obtenção da NH2PTriPP sem necessidade da separação da TPP. Após uma série de testes foi obtida a bisporfirina-bipiridina (BisPBipy) pela reação entre a NH2PTriPP e a DACBipy, sendo aspectos importantes nesta reação a obtenção do cloreto de ácido da bipiridina (DACBipy) in situ e utilização de excessos de aminoporfirina, ao invés de relações estequiométricas como é usual para este tipo de reação. Desta forma se evita a conversão do cloreto de ácido no respectivo ácido carboxílico que apresenta menor solubilidade e reatividade. Uma vez obtida a bisporfirina base livre foi possível sintetizar a respectiva bisporfirina metalada com íons de Zn(II) nas unidades de porlitina(BisZnPBipy) pela reação com cloreto de zinco. Os espectros de absorção da BisPBipy e BisZnPBipy apresentam perfil espectral característicos das porfirinas base livre e metaloporfirina, sendo os máximos de absorção tanto da banda Soret como das bandas Q praticamente coincidentes com os máximos de absorção da TPP e ZnTPP. As absortividades molares, entretanto, são duas vezes maiores, do que a TPP e ZnTPP, indicando a existência de duas unidades cromofóricas porfirínicas. Assim como observado para as propriedades de absorção UV-Vis, existe uma grande similaridade nas propriedades de emissão da BisPBipy e BisZnBipy com as moléculas modelo TPP e ZnTPP, respectivamente. A BisPBipy mantém o espectro de emissão de \"imagem especular reversa\" da TPP, indicando que a ligação a unidade de bipiridina não altera a geometria do estado excitado. Tanto a BisPBipy como a BisZnPBipy apresentam rendimentos quânticos de emissão de fluorescência similares a TPP e ZnTPP, indicando que para uma mesma fração de luz absorvida tem-se uma mesma emissão de fluorescência por unidade porfirínica presente nas bisporfirinas. Portanto as propriedades de absorção e emissão indicam que as unidades de porfirina atuam de forma independente não sendo influências pela ligação a unidade de bipiridina. Foi investigada a interação entre as bisporfirinas-bipiridinas e os íons Zn2+, Fe2+, Ni2+, Cu2+ e Co2+ em THF. Através de espectroscopia eletrônica UV-Vis e emissão de fluorescência foi possível constatar que ocorre interação entre as bisporfirinas-bipiridinas e os íons Ni2+, Cu2+ e Co2+. A presença destes íons metálicos em soluções das bisporfirinas-bipiridinas origina apenas pequenas alterações na intensidade da banda Soret; as bandas Q permanecem inalteradas. Este resultado é decorrente da interação dos íons metálicos com a unidade de bipiridina destas bisporfirinas, sendo que as alterações espectrais devidas a complexação ocorrem nas bandas centradas no ligante bipiridina que apresentam transições no ultravioleta, não interferindo apreciavelmente na região espectral das porfirinas. Contrariamente ao que foi observado nos espectros de absorção a presença de íons metálicos influencia de forma significativa a intensidade de fluorescência das bisporfirinas-bipiridinas investigadas, podendo ser observada a supressão de fluorescência em função do aumento da concentração dos íons metálicos. Utilizando os dados de emissão de fluorescência foi possível determinar a estequiometria dos complexos formados entre as bisporfirinas-bipiridinas e os íons metálicos. Co2+ e Cu2+ formam complexos de estequiometria 1:1, enquanto que Ni2+ forma complexos de estequiometria 2:1. A análise dos plots do tipo Stern-Volmer para a supressão de fluorescência das bisporfirinas-bipiridinas mostra um perfil exponencial típico de um processo de supressão não difusional. As constantes de Stern-Volmer aparentes (KSVap) mostram que não existe uma diferença significativa na eficiência de supressão independentemente da bisporfirina analisada ou do íon metálico supressor, sendo obtidos valores de KSVap de aproximadamente 5x105 M-1. Também foi realizado um estudo da interação das bisporfirinas-bipiridinas com o complexo [Eu(tta)3(H2O)2, não sendo observada uma interação apreciável nas condições normais de titulação, indicando que substituição das moléculas de água coordenada no complexo [Eu(tta)3(H2O)2 pela bipiridina não é favorecida em THF. Para as amostras submetidas a aquecimento é verificada uma supressão de fluorescência de aproximadamente 25-30%, indicando a ligação com a unidade bipiridina. Estas bisporfirinas se apresentam como moléculas promissoras no desenvolvimento de sistemas mais complexos contendo porfirinas devido a presença das unidades de bipiridina que possibilitam a formação de uma série de novas estruturas baseadas na coordenação a unidade de bipindina. Outra perspectiva é a elaboração bisporfirinas-bipiridinas, contendo outras metaloporfirinas como, por exemplo, de paládio, estanho, cobre, que também são luminescentes, porem com outras propriedades de estado excitado, resultando em sistemas de grande versatilidade. / In this work have been synthesized bisporphyrins held together by means of amide linkage at the 4,4\' position of 2,2-bipyridine, reacting the mono-functionalised aminophenyl-porphyrin (NH2PTriPP) and 4,4\'-diacid chloride bipyridine (DACBipy). A preliminary study was conducted for the establishment of the reaction conditions for the attainment of mono(nitrophenyl)-porphyrin (NO2PTriPP), the corresponding porphyrin precursor for the NH2PTriPP synthesis. The NO2PTriPP was obtained with yields in the range of 60% in nitration reactions of TPP in trifluoroacetic acid at room temperature, using twofold molar excess of sodium nitrite and period of reaction no longer than one minute. With these reaction conditions are achieved high reproducibility and the resulting mixture of NO2PTriPP and TPP can be directly reduced by tin chloride for obtaining NH2PTriPP without the need of TPP separation. After a series of trials the bisporphyrin-bipyridine (BisPBipy) was obtained reacting NH2PTriPP and DACBipy. Important features of this reaction conditions are the preparation in situ of the diacid chloride bipyridine and the use of a larger excess of aminoporphyrin than the usual for this kind of reaction. In this way, the formation of the corresponding carboxylic acid bipyridine is avoided. The correspondent Zn(II) bisporphyrin-bipyridine derivative (BisZnPBipy), was obtained reacting the free base BisPBipy with zinc chloride. The BisPBipy and BisZnPBipy show characteristic spectral profile of free base and metalloporphyrin spectra, and was observed a great similarity in the absorption maxima for the Soret and Q bands with the corresponding maxima of TPP and ZnTPP respectively. Otherwise the molar absorptivities are twofold higher, as an indication of the existence of two chromophoric units, as expected for bisporphyrins. As observed for the UV-Vis absorption spectra there are a great similarity in the emission properties of BisPBipy and BisZnPBipy and the models TPP and ZnTPP respectively. The \"reverse mirror image\" emission of TPP is also observed for BisPBipy, indicating that the porphyrin chromophore bounded to the bipyridine unit does not alter the excited state geometry. The fluorescence quantum yields for BisPBipy and BisZnPBipy are the same to the observed for TPP and ZnTPP, indicating that for the same fraction of absorbed light the same fluorescence is displayed per porphyrin unit in the bisporphyrin. It can be concluded from the absorption and emission properties that the porphyrin chromophore in these bisporphyrins behaves as independent units. The interaction between the bisporphyrins-bipyridines and Zn2+, Fe2+, Ni2+ , Cu2+ and Co2+ ions in THF was investigated by means of UV-Vis electronic absorption and fluorescence emission spectroscopies, the results show that the interaction occurs only with Ni2+ , Cu2+ e Co2+ without formation of any precipitate. The presence of these metal ions in the bisporphyrin-bipyridine solutions causes small decrease in the intensity of the Soret band; the Q bands remaining unaltered. These spectral features are due to the interaction at the bipyridine unit of these bisporphyrins, and the resulting spectral changes occur in the ligand-centered bipyridine bands showing absorption in the ultra-violet, not matching appreciably the porphyrin absorption spectrum region. The presence of metal ions deeply influences the fluorescence emission intensity of these bisporphyrins, being observed the fluorescence quenching as function of the increasing metal ion concentration. From the fluorescence emission data the stoichiometry of the complexes formed in solution has been estimated for Co2+ and Cu2+ as 1:1 and for Ni2+ as 2:1. The analysis of the Stern-Volmer plots shows a typical profile of a non diffusional quenching process. The apparent Stern-Volmer constants (KSVap) show no significant differences independently of the bisporphyrin or the metal ion employed, being obtained KSVap values of approximately 5x105 M-1. In addition, it was studied the interaction between the bisporphyrins-bipyridines and the [Eu(tta)3(H2O)2 complex, with no appreciable interaction occurring at normal titrating conditions, as an indicative of disfavoring process of the replacement of the coordinated water molecules by the bipyridine ligand in THF. For the samples submitted to heating a fluorescence quenching of 25-30% can be observed, indicating that under heating conditions the complex with the bipyridine is formed. These bisporphyrins are promising in the development of more complex systems containing porphyrins due the presence of the bipyridine units that enable the formation of a series of new structures based on the bipyridine coordination. Another perspective is regarded to the elaboration of bisporphyrins-bipyridine containing different metalloporphyrins, such as palladium, tin, copper, that show luminescence, but with different excited state properties than the zinc porphyrin ones, resulting in systems of great versatility.

<p>Degradation of the mRNA 3'-end located poly(A) tail is an important step for mRNA decay in mammalian cells. Thus, to understand mRNA decay in detail, it is important to identify the catalytic activities involved in degrading poly(A). We identified and purified a 54-kDa polypeptide responsible for poly(A)-specific 3' exonuclease activity in calf thymus extracts. The 54-kDa polypeptide is a proteolytic fragment of the poly(A)-specific ribonuclease (PARN) 74-kDa polypeptide. PARN is a divalent metal ion dependent, poly(A)-specific, oligomeric, processive and cap interacting 3' exonuclease. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. The interaction with the 5'-end cap structure stimulates PARN activity and also amplifies the processivity of the deadenylation reaction. Furthermore, the cap binding site and the active site of PARN are separate from each other. To characterise the active site of PARN, we per-formed side-directed mutagenesis, Fe²⁺-mediated hydroxyl radical cleavage and metal ion switch experiments. We have demonstrated that the conserved acidic amino acid residues D28, E30, D292 and D382 of human PARN are essential for PARN activity and that these amino acid residues are directly involved in the co-ordination of at least two metal ions in the active site of PARN. Phosphorothioate modification on RNA substrates revealed that the pro-R oxygen atom of the scissile phosphate group interacts directly with the metal ion(s). Based on our studies, we propose a model for the action of PARN. Similarly to what has been observed for ribozymes, aminoglycoside antibiotics inhibit PARN activity, most likely by the displacement of catalytically important divalent metal ions. Among the aminoglycoside antibiotics tested, neomycin B is the most potent inhibitor. We speculate that inhibition of enzymes using similar catalytic mechanisms as PARN could be a reason for the toxic side effects caused by aminoglycoside antibiotics in clinical practice. </p>

<p>Metal ions solvated in aqueous, non-aqueous, and mixtures of solvents occur in many chemical contexts, for example in electrochemical applications and solvent separation. Solvated ions appear in high concentration in the living organisms, where their presence or absence can fundamentally alter the functions of life. In many of these cases, understanding the selective solvation and the dynamics of the ions is essential for the understanding of the processes involved.</p><p>Computer simulation provides a molecular level of detail of the solvation process usually not available from experiments. The quality of the interaction models employed in the theoretical description is of particular importance, since even rather small changes in the interaction can lead to substantial and qualitative differences.</p><p>This thesis describes the development of a sequence of increasingly refined analytical ion-solvent potentials from <i>ab initio</i> calculations for the systems Li⁺(<i>aq</i>), Na⁺(<i>aq</i>), Mg²⁺(<i>aq</i>), Al³⁺(<i>aq</i>), Li⁺(<i>MeCN</i>), Na⁺(<i>MeCN</i>), Li⁺(<i>aq, MeCN</i>), and Na⁺(<i>aq, MeCN</i>). Molecular dynamics simulations using these potentials were subsequently performed, and some key-properties computed. The reliability of the computed thermodynamical, structural and dynamical properties was scrutinized.</p>

<p>The aqueous solvation of metal ions is one of the long-standing and complex problems in chemistry, with implications for and applications in a broad range of biochemical and electrochemical systems, where water is the all-pervasive medium.</p><p>This thesis describes computer simulations of Al³⁺(<i>aq</i>), Fe²⁺(<i>aq</i>), Fe³⁺(<i>aq</i>) and Cu²⁺(<i>aq</i>). Various aspects of the solvation of these polyvalent metal ions in water are addressed, at different levels of theory, using Car-Parrinello molecular dynamics, classical molecular dynamics and quantum-mechanical cluster calculations. Polyvalent metal ions are particularly interesting because of their large influence on the solvent structure, dynamics and thermodynamics, as well as on the properties of the individual solvent molecules. Polyvalent metal ions in aqueous solution also constitute a challenging subject for computer simulations since a sophisticated interaction model is needed to incorporate the large many-body effects. </p><p>All the ion-water coordination figures in this thesis are octahedral, except in the Cu²⁺(<i>aq</i>) solution, where the ion is penta-coordinated with four equatorial neighbours in a plane and one axial neighbour located ~0.45 Å further out from the ion. The equatorial ion-water bonds have covalent character, while the axial water molecule is only electrostatically bound. For all the ions, the OD stretching frequencies of the first-shell water molecules are much more downshifted than in liquid water. In the case of Cu²⁺(<i>aq</i>), however, only the OD frequencies of the equatorial water molecules are downshifted with respect to bulk water whereas the OD frequencies of the axial water molecule are slightly upshifted. </p><p>Various limitations of the Car-Parrinello molecular dynamics simulations have been explored and compared, such as finite system-size effects and shortcomings in the electronic structure calculations. The Car-Parrinello simulations are found to give reasonable descriptions of the polyvalent metal ions in aqueous solution.</p>

The aqueous solvation of metal ions is one of the long-standing and complex problems in chemistry, with implications for and applications in a broad range of biochemical and electrochemical systems, where water is the all-pervasive medium. This thesis describes computer simulations of Al3+(aq), Fe2+(aq), Fe3+(aq) and Cu2+(aq). Various aspects of the solvation of these polyvalent metal ions in water are addressed, at different levels of theory, using Car-Parrinello molecular dynamics, classical molecular dynamics and quantum-mechanical cluster calculations. Polyvalent metal ions are particularly interesting because of their large influence on the solvent structure, dynamics and thermodynamics, as well as on the properties of the individual solvent molecules. Polyvalent metal ions in aqueous solution also constitute a challenging subject for computer simulations since a sophisticated interaction model is needed to incorporate the large many-body effects. All the ion-water coordination figures in this thesis are octahedral, except in the Cu2+(aq) solution, where the ion is penta-coordinated with four equatorial neighbours in a plane and one axial neighbour located ~0.45 Å further out from the ion. The equatorial ion-water bonds have covalent character, while the axial water molecule is only electrostatically bound. For all the ions, the OD stretching frequencies of the first-shell water molecules are much more downshifted than in liquid water. In the case of Cu2+(aq), however, only the OD frequencies of the equatorial water molecules are downshifted with respect to bulk water whereas the OD frequencies of the axial water molecule are slightly upshifted. Various limitations of the Car-Parrinello molecular dynamics simulations have been explored and compared, such as finite system-size effects and shortcomings in the electronic structure calculations. The Car-Parrinello simulations are found to give reasonable descriptions of the polyvalent metal ions in aqueous solution.

Metal ions solvated in aqueous, non-aqueous, and mixtures of solvents occur in many chemical contexts, for example in electrochemical applications and solvent separation. Solvated ions appear in high concentration in the living organisms, where their presence or absence can fundamentally alter the functions of life. In many of these cases, understanding the selective solvation and the dynamics of the ions is essential for the understanding of the processes involved. Computer simulation provides a molecular level of detail of the solvation process usually not available from experiments. The quality of the interaction models employed in the theoretical description is of particular importance, since even rather small changes in the interaction can lead to substantial and qualitative differences. This thesis describes the development of a sequence of increasingly refined analytical ion-solvent potentials from ab initio calculations for the systems Li+(aq), Na+(aq), Mg2+(aq), Al3+(aq), Li+(MeCN), Na+(MeCN), Li+(aq, MeCN), and Na+(aq, MeCN). Molecular dynamics simulations using these potentials were subsequently performed, and some key-properties computed. The reliability of the computed thermodynamical, structural and dynamical properties was scrutinized.

Degradation of the mRNA 3'-end located poly(A) tail is an important step for mRNA decay in mammalian cells. Thus, to understand mRNA decay in detail, it is important to identify the catalytic activities involved in degrading poly(A). We identified and purified a 54-kDa polypeptide responsible for poly(A)-specific 3' exonuclease activity in calf thymus extracts. The 54-kDa polypeptide is a proteolytic fragment of the poly(A)-specific ribonuclease (PARN) 74-kDa polypeptide. PARN is a divalent metal ion dependent, poly(A)-specific, oligomeric, processive and cap interacting 3' exonuclease. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. The interaction with the 5'-end cap structure stimulates PARN activity and also amplifies the processivity of the deadenylation reaction. Furthermore, the cap binding site and the active site of PARN are separate from each other. To characterise the active site of PARN, we per-formed side-directed mutagenesis, Fe2+-mediated hydroxyl radical cleavage and metal ion switch experiments. We have demonstrated that the conserved acidic amino acid residues D28, E30, D292 and D382 of human PARN are essential for PARN activity and that these amino acid residues are directly involved in the co-ordination of at least two metal ions in the active site of PARN. Phosphorothioate modification on RNA substrates revealed that the pro-R oxygen atom of the scissile phosphate group interacts directly with the metal ion(s). Based on our studies, we propose a model for the action of PARN. Similarly to what has been observed for ribozymes, aminoglycoside antibiotics inhibit PARN activity, most likely by the displacement of catalytically important divalent metal ions. Among the aminoglycoside antibiotics tested, neomycin B is the most potent inhibitor. We speculate that inhibition of enzymes using similar catalytic mechanisms as PARN could be a reason for the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

The present thesis entitled, "Molecular expression through fluorescence: Studies in probe design and aggregation" describes very simple bi-functional donor-acceptor poly-aromatic fluorophores that have been shown to possess distinctive properties depending on the context in which they are studied. In a sense, this work is an effort in exemplifying the inherent diversity and power of "molecular expression", with the central theme here being the phenomenon of fluorescence. The work has been divided into four chapters, each having a self-contained introduction. Chapter 1: First instance of metal ion (Zn2+) sensing exclusively at amphiphilic interfaces. (1 -pyrenyl)rnethyl-bis- [(2-pyridyl)methyl]amine (Pybpa), a simple, bi-functional fluorophore was synthesized. Pybpa has the modular design of a photoinduced electron transfer (PET) based analyte sensor. In Pybpa, a photoinduced electron transfer (PET) operates from the pyrenyl nitrogen (PyCH2-iV) to the excited pyrenyl (Py*) chromophore leading to fluorescence quenching. Zn2+ ion binding to the bis-picolyl (bpa) unit of Pybpa stops the PET process and leads to fluorescence enhancement. Thus Pybpa was able to sense Zn2+" in organic solvents. In water, however, Pybpa showed pronounced aggregation and the probe did not sense any metal ion. Surfactant micelles provide hydrophobic regions in water and the dynamic rnicellar assemblies could disrupt Pybpa aggregates. Pybpa monomers solubilized in micelles were responsive to Zn2+ in the low micro molar concentration range. The metal ion sensing on micelles was reflective of the charge of the interface. The sensing is negligible on cationic surface (CTAB), moderate on negatively charged surface (SDS micelles) and is the most efficient on neutral interface provided by TWEEN-20 micelles. With the Pybpa 'sensor*, no sensing is possible in water and hence the sensing is exclusive to the interface. Pybpa doped in membranous aggregates like phosphatidylcholine (PC) lipid bilayers, exists in monomeric form, and was able to sense Zn . The sensing on phosphatidylcholine (PC) bilayer vesicles was found to depend on the fluidity of the membrane. Zn2^ sensing with interfacially bound probe "was extended to a globular protein bovine serum albumin (BSA). BSA, a carrier protein, can bind hydrophobic molecules as well as metal ions like Zn2f. BSA was shown to disrupt Pybpa aggregation and bind Pybpa in a facile manner. BSA bound Pybpa was able to sense externally added Zn2+. Biological sensing of trace amounts of Zn2+ has been considered important since Zn2+ is crucial for eukaryotic systems. This is the first example of such 'exclusive' interfacial sensing of a metal ion. Chapter 2: Towards understanding and modulating self-assembly of pyrenyl bis-picolyl a mine: Organic nanoparticles that show tunable emission. Pybpa was found to aggregate in water in the size range of 80-250 nm. Evidence of aggregation was seen at concentrations as low as 1 |iM. The nanoscopic particles formed were characterized through transmission electron microscopy (TEM) and dynamic light scattering (DLS). Pybpa in water showed dual emission bands, with one band resernhling the emission from 'monomeric' Pybpa (as seen in solutions in organic solvents) and a broad red-shifted emission band (A,max ~ 480 ran) designated as "aggregate/nanoparticle" emission. Distinct excitation spectra for the two emission bands indicate that the bands (the '390 nm' band and the '480 nm' band) originate through distinct excitation/emission channels. The time resolved emission decay for the 'monomer' emission (397 nm) showed a substantial contribution from a long-lived pyrene-like excited state (x = 103.9 ns, 40% relative amplitude). On the other hand, the decay at 475 nm (for the nanoparticle/aggregate emission band) was considerably faster, with no evidence of any pyrene-like long-lived state. The short lifetimes indicated an exciplex nature of the red-shifted emission band, X-,nax~480 nm. The effect of temperature and urea on these aggregates was examined. The nanoparticles formed even in a concentrated urea solution (7.8 M). The aggregates formed in urea were found to be more emissive, indicating a 'looser' aggregate with reduced fluorescence quenching. Similar results were obtained on heating the aggregate. Increasing the concentration of Pybpa in water causes a change in the nature of the colloids formed as exemplified by increase in aggregate size and a decrease in the polydispersity index. Also seen was a substantial red shift in the 'aggregate emission'. At higher concentrations, the presence of three independent excitation/emission channels was observed. It is likely that a new type of aggregated Pybpa species formed at higher concentration, which emits at longer wavelength (A,rnax~540 nm), In such a scenario, it is possible to tune the emission wavelength by the choice of appropriate wavelength of excitation. Further, there is an opportunity to tailor the emission properties by controlling the aggregation behavior. The modulation of emission is one of the primary goals of research on fluorescent organic nanoparticles. Chapter 3: Photophysical properties of aryl-terpyridines in solution, solid and aggregated state: Unique CT emission from nanoparticles in water. Two aryl terpyridines, 4T-(l-pyrenyl)-2,2l:6'52fl-terpyridine (Pytpy) and 4'-(9-anthryl)-2,2':6',2n-terpyridine (Antpy), where the fluorophoric pyrene or anthracene unit is directly coupled to the terpyridine unit, were synthesized. The aryl terpyridines conjugates can be viewed as donor-acceptor molecules that are conformationally labile, with the possibility of rotation around three single bonds. It was of interest to see as to how conformational effects express themselves in different environments, especially in relation to the possibility of charge separation. Crystal structure data and Serni-empirical AMI calculations revealed a twisted molecular conformation for each of the molecules. Absorption and emission (steady state as well as tirne-resolved) behavior of Pytpy and Antpy in various organic solvents have been presented. The molecules showed only limited conjugation between the two units in the absorption behavior with the degree of conjugation being greater for Pytpy. In the emission behavior, only a single emission band (with a single lifetime) was observed in all organic solvent. Steady state and time resolved fluorescence data suggest the existence of a mixed or coupled, largely 7t—7i* state, with only marginal charge separation. The various photophysical parameters have been determined for the two systems. It appears that in the excited state, the inesomeric interactions show an increase for each of the two aryl-terpyridines, indicating at least a partially planar geometry in the excited state. Some specific solvent effects were observed for the molecules in alcoholic solvents and there was evidence of excited state H-bonding occurs for the aryl terpyridines in polar protic organic solvents, especially methanol. Pytpy and Antpy self-assembled in water over a large concentration range (1-100 |xM) to form spherical nanoparticles in the size range of 150-200 nm, as characterized by TEM and DLS. The absorption spectra for both conjugates showed red shift of the absorption bands in water (-10 nrn) along with significant tailing of the long-wavelength bands. The change in emission behavior in going from solution to the aggregates in water was very dramatic. Multiple, broadened, highly red-shifted emission bands for both Antpy and Pytpy were observed. Quite significantly, a long lifetime component in the emission decay was shown by the conjugates in water as compared to the lifetimes observed in solution. The data points towards a unique CT emission for Antpy and Pytpy aggregates in water. The excitation spectra for the multiple emission bands seen for Pytpy (or Antpy) were observed to be identical. Thus a single ground state population is responsible for emission over the entire range (approximately 420 nin - 600 nm). The existence of multiple emission bands and the large bathochromic shifts are exclusively due to excited state effects in the aggregated state in water. It appears that excited state H-bonding of the tpy N with water helps facilitate the excited state CT. The solid-state behavior of Pytpy and Antpy lias been examined and the emission from the two crystalline solids is very distinct. Antpy emission showed a X,,nax at -430 nm while Pytpy emission peaked at ~ 560 nm. The difference in the solid-state emission behavior exhibited by Pytpy and Antpy is explained through a consideration of the crystal packing for the two molecules. The degree of n-facial stacking was observed to be much greater for Pytpy. The observation of the distinct packing and emission shown by solid Pytpy and Antpy is highly significant if one considers the identical emission shown by the aqueous nanoparticles of the two molecules and brings to fore the 'nanoparticle effect' in water as compared to a simple concentration effect. It was also demonstrated that it was possible to modulate the aggregation of the terpyridines through additives, like metal ions Chapter 4: Pyrenyl terpyridine as a ratiometric fluorescence probe for sensing order and polarity of membranous aggregates. Pytpy was examined for its utility in probing surfactant aggregates, particularly membranous assemblies. la lipid bilayer vesicles made of phosphatidylcholine (PC) lipids (like dimyristoyl phosphatidylcholine, DMPC or egg-yolk PC) Pytpy showed an emission profile with marked similarity to that shown by the probe in water. Specifically, a broad red-shifted emission with A,maxin. the 500 nm region was observed. In addition, a peak in the -420 nm region was also seen. Fluorescence anisotropy was used to confirm the presence of vesicle-bound probe. Excitation spectra confirmed the presence of two distinct probe populations, om responsible for the '420 nm9 emission and another population responsible for the multiple, red-shifted emission bands. The emission behavior was indicative of aggregation of Pytpy on the vesicle surface and CT effects operating in conjunction with H-bonding. Fluorescence lifetime measurements, carried out at different Is suggest the CT nature of the red-shifted emission. The aggregation of the probe on the bilayer interface was confirmed by concentration and temperature dependence of the emission profile. The role of water in stabilizing this CT emission on bilayer surfaces was shown with use of a surface dehydrating agent polyethylene glycol (PEG). All these results helped build a model for the behavior of Pytpy in water. Pytpy aggregates on bilayer surface and shows a red-shifted CT emission with stabilization by interfacial water. Thus, the Pytpy 'aggregate' has a shallow, water accessible location in the bilayer. In addition to this, there is another Pytpy population responsible for the emission in the 420 nm region, and this second population might have a comparatively deeper location. The wavelength of the CT emission was sensitive to the polarity of the interface as evidenced "by the results obtained with bilayers made of a number of PC lipids. In general, the X™ax of the CTband showed a red shift with increasing polarity. The increase in polarity also caused an increase in the average lifetime of the probe. Pytpy could distinguish between vesicles made of lipids of different head groups. Aggregates made of phosphatidylethanolamine (PE) head group are in general less hydrated than PC lipid assemblies and Pytpy emission reflected this when examined in vesicles made of related lipids (dioleoyl lipids, DOPC and DOPE; dirnyristoyl lipids, DMPC and DMPE). Pytpy emission from PE vesicles was quenched and showed a pronounced blue shift in the emission Xmax vis-a-vis PC bilayers. Thus, dehydration of the interface consistently led to the destabilization of the CT state. Further, Pytpy emission was also responsive to hydration in more complex mixed PC-PE assemblies. Pytpy emission "behavior was also used to probe fluidity in complex "mixed" lipid assemblies- The effect of cholesterol on DMPC bilayers in terms of its known ability to dehydrate the bilayer was reported through a blue-shift Xmax of CT emission band. Further, cholesterol also causes drastic change in the bilayer at concentrations greater than ~ 30 mol%. This change in the bilayer was sensed through a sudden reduction in fluorescence intensity. Also from a careful analysis of Pytpy in various PC and PE vesicles, it emerged that the more fluid aggregates showed larger quantum yields. Thus, Pytpy could simultaneously report on both the polarity and fluidity of lipidic aggregates. Pytpy could also provide information about the order of an assembly. While the probe aggregated in bilayers and other membranous assemblies and showed water assisted CT emission, in more dynamic assemblies like micelles, Pytpy aggregates were not sustained, Pytpy in micelles showed emission spectra very similar to that seen in solutions in aprotic organic solvents. Thus, Pytpy proved to be a very useful ratiometric sensor for vesicle-to-rnicelle transition. Also, it has been possible to study some surfactant-lipid mixed assemblies that show phase separation. Pytpy reported the formation of a 'rigid', bilayer-like phases in mixed assemblies that are called bicelles.

Abstract Starch is one of the most abundant polysaccharides found in nature and is widely utilized in various fields of industry. Due to the complex structure of native starch it is insoluble in most organic solvents and needs modification prior utilization. In this study, ionic liquids (ILs), modern green chemistry alternatives for common solvents were used as reaction media in starch modification. At first various starch species were depolymerized in 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) with p-TsOH as a catalyst. Microwave activation or conventional bath heating were used as heating methods while HPLC-ELSD was used as an analytical method. All studied starch species depolymerized similarly into water-soluble starch oligomers while microwave activation shortened the depolymerization time considerably compared to oil bath heating. Barley starch was chosen for further experiments, in which various ILs were studied as potential media for starch dissolution and depolymerization. Results suggested that both the anion and the cation part of the IL had an effect on the dissolution and depolymerization of barley starch. After the depolymerization reactions, the depolymerized barley starch was further modified by cationization. [AMIM]Cl was used as the reaction media, microwave activation as the heating method while HPLC-ELSD, 1H NMR and elemental analysis were used as analytical methods. The modified products had DS values from 0.2 to 0.5 depending on the reaction conditions. The products were studied as potential binding agents for heavy metal ions which showed that moderately substituted modified starch (DS 0.4) could be used to bind Cu(II), Fe(III) and Zn(II) ions from an aqueous solution. Finally, potato peel waste was studied as an alternative starch source to produce cationized starch for wastewater purification. Peel waste was pre-treated by alkaline depolymerization after which it was cationized in a water solution to produce cationized products with DS from 0 to 0.35. The cationized peel waste products were studied preliminary as binding agents for Cu(II) ions from a water solution using ICP-OES as an analytical method. The results suggested that when the molar ratio between cationized waste starch and copper was 3:1, cationized waste starch was an effective binding agent for copper ions. / Tiivistelmä Tärkkelys on yksi yleisimmistä luonnossa esiintyvistä polysakkarideista. Sitä hyödynnetään useilla eri teollisuuden aloilla. Monimutkaisen rakenteensa vuoksi tärkkelys on liukenematon useimpiin orgaanisiin liuottimiin ja veteen, minkä vuoksi sitä täytyy modifioida ennen käyttöä. Tässä väitöstutkimuksessa tärkkelyksen modifioinnissa käytettiin ionisia nesteitä reaktioväliaineena. Tutkimuksen alussa eri tärkkelyslajeja depolymeroitiin 1-allyyli-3-metyyli-imidatsoliumkloridissa ([AMIM]Cl) katalyyttinä p-TsOH. Mikroaaltoaktivointia ja haudekuumennusta käytettiin vaihtoehtoisina lämmitysmenetelminä. Reaktion edistymistä ja tuotteiden muodostumista tutkittiin HPLC-ELSD -menetelmällä. Eri tärkkelyslajit depolymeroituivat samankaltaisesti vesiliukoisiksi, lyhytketjuisiksi tärkkelysoligomeereiksi. Mikroaaltoaktivointi lyhensi reaktioaikaa haudekuumennukseen verrattuna. Tutkimuksen seuraavassa vaiheessa tutkittiin ohratärkkelyksen liukoisuutta ja depolymeroitumista eri ionisissa nesteissä. Tulosten perusteella ionisen nesteen sekä anioni- että kationiosa vaikuttivat tärkkelyksen liukenemiseen. Depolymeroidun ohratärkkelyksen modifiointitutkimuksia jatkettiin [AMIM]Cl:ssa kationisoinnilla. Lämmitysmenetelmänä käytettiin mikroaaltoaktivointia. Tuotteet tutkittiin käyttäen alkuaineanalyysiä sekä HPLC-ELSD- että 1H NMR-tekniikoita. Kationisoitujen tuotteiden substituutioaste (DS) vaihteli reaktio-olosuhteista riippuen välillä 0.2–0.5. Saatuja tuotteita tutkittiin raskasmetalli-ionien sitomisessa vesiliuoksesta. Havaittiin, että kohtalaisesti substituoitu (DS 0.4) modifioitu tärkkelys sitoi Cu(II)-, Fe(III)- ja Zn(II)-ioneja vesiliuoksesta. Tutkimuksen loppuosassa tutkittiin perunan kuorijätettä vaihtoehtoisena tärkkelyslähteenä kationisoidun tärkkelyksen valmistamisessa. Kuorijäte esikäsiteltiin kuumentamalla se emäksisessä etanoliliuoksessa, minkä jälkeen sille suoritettiin kationisointi vesiliuoksessa. Kationisten tuotteiden substituutioasteet vaihtelivat välillä 0–0.35. Tuotteiden soveltuvuutta Cu(II)-ionien sitomiseen vesiliuoksesta tutkittiin ICP-OES -menetelmän avulla. Alustavien tulosten mukaan kationisoitu jätetärkkelys sitoi kupari-ioneja vedestä, kun tärkkelyksen ja kuparin moolisuhde oli 3:1.